Bridging molecular to cellular scales for models of membrane receptor signaling.

Biochemical interactions at membranes are the starting points for cell signaling networks. But bimolecular reaction kinetics are difficult to experimentally measure on 2-dimensional membranes and are usually measured in volumetric assays. Membrane tethering produces confinement and steric effects that will significantly impact binding rates in ways that are not readily estimated from volumetric measurements.

Measurement of solubility product reveals the interplay of oligomerization and self-association for defining condensate formation.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation is daunting. Using experiments and computation, we therefore studied a simple model system consisting of polySH3 and polyPRM designed for pentavalent heterotypic binding.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes.

Measurement of solubility product in a model condensate reveals the interplay of small oligomerization and self-association.

Cellular condensates often consist of 10s to 100s of distinct interacting molecular species. Because of the complexity of these interactions, predicting the point at which they will undergo phase separation into discrete compartments is daunting. Using experiments and computation, we therefore studied a simple model system consisting of 2 proteins, polySH3 and polyPRM, designed for pentavalent heterotypic binding.

Di-5-ANEQ(F)PTEA Offers Better Performance than Di-4-ANEQ(F)PTEA for In-Situ Cardiac Optical Mapping.

Cardiac optical mapping has traditionally been performed in ex-vivo, motion-arrested hearts. Recently, in-situ cardiac optical mapping has been made possible by both motion correction techniques and long-wavelength voltage sensitive dyes (VSDs). However, VSDs have been observed to wash out quickly from blood-perfused in-situ hearts.

Optical mapping of cardiac electromechanics in beating in vivo hearts.

Optical mapping has been widely used in the study of cardiac electrophysiology in motion-arrested, ex vivo heart preparations. Recent developments in motion artifact mitigation techniques have made it possible to optically map beating ex vivo hearts, enabling the study of cardiac electromechanics using optical mapping. However, the ex vivo setting imposes limitations on optical mapping such as altered metabolic states, oversimplified mechanical loads, and the absence of neurohormonal regulation.

Recent advances and current limitations of available technology to optically manipulate and observe cardiac electrophysiology.

Optogenetics, utilising light-reactive proteins to manipulate tissue activity, are a relatively novel approach in the field of cardiac electrophysiology. We here provide an overview of light-activated transmembrane channels (optogenetic actuators) currently applied in strategies to modulate cardiac activity, as well as newly developed variants yet to be implemented in the heart. In addition, we touch upon genetically encoded indicators (optogenetic sensors) and fluorescent dyes to monitor tissue activity, including cardiac transmembrane potential and ion homeostasis.

Near-infrared voltage-sensitive dyes based on chromene donor.

Voltage-sensitive dyes (VSDs) are used to image electrical activity in cells and tissues with submillisecond time resolution. Most of these fast sensors are constructed from push-pull chromophores whose fluorescence spectra are modulated by the electric field across the cell membrane. It was found that the substitution of naphthalene with chromene produces a 60 to 80 nm red-shift in absorption and emission spectra while maintaining fluorescence quantum efficiency and voltage sensitivity.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value.

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart.

Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem.

Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments.

MolClustPy: a Python package to characterize multivalent biomolecular clusters.

Summary: Low-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become supply-limited large clusters. In stochastic simulations, such clusters display a wide range of sizes and compositions. We have developed a Python package, MolClustPy, which performs multiple stochastic simulation runs using NFsim (Network-Free stochastic simulator); MolClustPy characterizes and visualizes the distribution of cluster sizes, molecular composition, and bonds across molecular clusters.

Optogenetic manipulation of cardiac repolarization gradients using sub-threshold illumination.

Mechanisms underlying cardiac arrhythmias are typically driven by abnormalities in cardiac conduction and/or heterogeneities in repolarization time (RT) across the heart. While conduction slowing can be caused by either electrophysiological defects or physical blockade in cardiac tissue, RT heterogeneities are mainly related to action potential (AP) prolongation or abbreviation in specific areas of the heart. Importantly, the size of the area with altered RT and the difference between the short RT and long RT (RT gradient) have been identified as critical determinators of arrhythmogenicity.

MolClustPy: A Python Package to Characterize Multivalent Biomolecular Clusters.

S Ummary: Low-affinity interactions among multivalent biomolecules may lead to the formation of molecular complexes that undergo phase transitions to become extra-large clusters. Characterizing the physical properties of these clusters is important in recent biophysical research. Due to weak interactions such clusters are highly stochastic, demonstrating a wide range of sizes and compositions.

The maximum solubility product marks the threshold for condensation of multivalent biomolecules.

Clustering of weakly interacting multivalent biomolecules underlies the formation of membraneless compartments known as condensates. As opposed to single-component (homotypic) systems, the concentration dependence of multicomponent (heterotypic) condensate formation is not well understood. We previously proposed the solubility product (SP), the product of monomer concentrations in the dilute phase, as a tool for understanding the concentration dependence of multicomponent systems.

Fast Optical Investigation of Cardiac Electrophysiology by Parallel Detection in Multiwell Plates.

Current techniques for fast characterization of cardiac electrophysiology employ optical technologies to control and monitor action potential features of single cells or cellular monolayers placed in multiwell plates. High-speed investigation capacities are commonly achieved by serially analyzing well after well employing fully automated fluorescence microscopes. Here, we describe an alternative cost-effective optical approach (MULTIPLE) that exploits high-power LED arrays to globally illuminate a culture plate and an sCMOS sensor for parallel detection of the fluorescence coming from multiple wells.

Hyperexcitable Phenotypes in Induced Pluripotent Stem Cell-Derived Neurons From Patients With 15q11-q13 Duplication Syndrome, a Genetic Form of Autism.

Background: Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, patients with Dup15q commonly meet the criteria for autism spectrum disorder and have a high prevalence of seizures. It is known from mouse models that synaptic impairments are a strong component of Dup15q pathophysiology; however, cellular phenotypes that relate to seizures are less clear.

Mechanism of actin filament nucleation.

We used computational methods to analyze the mechanism of actin filament nucleation. We assumed a pathway where monomers form dimers, trimers, and tetramers that then elongate to form filaments but also considered other pathways. We aimed to identify the rate constants for these reactions that best fit experimental measurements of polymerization time courses.

A Complete and Low-Cost Cardiac Optical Mapping System in Translational Animal Models.

Clinicians, biologists, physicists, engineers, and computer scientists are coming together to better understand heart disease, which is currently the leading cause of death globally. Optical mapping, a high-speed fluorescence imaging technique that visualizes and measures key cardiac parameters such as action potentials, cytosolic calcium transients, and fibrillation dynamics, is a core research tool that has arisen from such interdisciplinary collaborations. In an effort to broaden its use, especially among clinical scientists and students, we developed a complete and low-cost optical mapping system, including a constant-flow Langendorff perfusion system, which minimizes the economic threshold to widespread use of this powerful tool in cardiac electrophysiology research.

The solubility product extends the buffering concept to heterotypic biomolecular condensates.

Biomolecular condensates are formed by liquid-liquid phase separation (LLPS) of multivalent molecules. LLPS from a single ("homotypic") constituent is governed by buffering: above a threshold, free monomer concentration is clamped, with all added molecules entering the condensed phase. However, both experiment and theory demonstrate that buffering fails for the concentration dependence of multicomponent ("heterotypic") LLPS.

Ten steps to investigate a cellular system with mathematical modeling.

Cellular and intracellular processes are inherently complex due to the large number of components and interactions, which are often nonlinear and occur at different spatiotemporal scales. Because of this complexity, mathematical modeling is increasingly used to simulate such systems and perform experiments in silico, many orders of magnitude faster than real experiments and often at a higher spatiotemporal resolution. In this article, we will focus on the generic modeling process and illustrate it with an example model of membrane lipid turnover.

Recent progress in optical voltage-sensor technology and applications to cardiac research: from single cells to whole hearts.

The first workshop on Novel Optics-based approaches for Cardiac Electrophysiology (NOtiCE) was held in Florence Italy in 2018. Here, we learned how optical approaches have shaped our basic understanding of cardiac electrophysiology and how new technologies and approaches are being developed and validated to advance the field. Several technologies are being developed that may one day allow for new clinical approaches for diagnosing cardiac disorders and possibly intervening to treat human patients.

Transcranial Recording of Electrophysiological Neural Activity in the Rodent Brain Using Functional Photoacoustic Imaging of Near-Infrared Voltage-Sensitive Dye.

Minimally-invasive monitoring of electrophysiological neural activities in real-time-that enables quantification of neural functions without a need for invasive craniotomy and the longer time constants of fMRI and PET-presents a very challenging yet significant task for neuroimaging. In this paper, we present functional PA (fPA) imaging of chemoconvulsant rat seizure model with intact scalp using a fluorescence quenching-based cyanine voltage-sensitive dye (VSD) characterized by a lipid vesicle model mimicking different levels of membrane potential variation. The framework also involves use of a near-infrared VSD delivered through the blood-brain barrier (BBB), opened by pharmacological modulation of adenosine receptor signaling.